De Novo Protein Design Overcomes Gene Editing Limits to Sustain CAR-T and TCR-T Cell Function

Key Takeaways

• De novo designed proteins bind and modulate entire target families simultaneously, bypassing the translational hurdles of multi-allele gene editing.
• The OUTLAST Regulator platform employs three distinct functional strategies, Designed Degraders, Transcription Factor Modulators, and Common Substrate Modulators, to rewire complex cell fate decisions.
• Pan-NR4A OUTLAST Regulators significantly enhance CAR-T and TCR-T cell functional persistence and tumor killing in rigorous solid tumor models both in vitro and in vivo.

In a recent preprint, researchers from Outpace Bio and the University of Washington present “OUTLAST Regulators,” de novo designed, compact protein modules (<1 kb DNA) that simultaneously modulate multiple target proteins to reprogram cellular pathways and sustain T cell functional persistence. 

Multi-target protein design strategies overcome traditional gene editing limitations

Solid tumors present a hostile microenvironment that redundantly drives chimeric antigen receptor (CAR) and transgenic T cell receptor (TCR) T cells into a state of dysfunction. Conventional approaches to counteract cell therapy dysfunction typically rely on single-gene knockouts or static gene overexpression, which fail to address these multifaceted pathways and face severe vector payload limits during multi-allele editing. 

The authors focused on target families known to drive T cell dysfunction: the nuclear receptor subfamily 4A (NR4A) transcription factors, the Casitas B-lineage lymphoma (Cbl) E3 ligases Cbl-b and c-Cbl, and the Suppressor of Cytokine Signaling (SOCS) family. Rather than knocking out individual genes, three distinct classes of compact protein regulators were computationally built using tools like RFdiffusion and ProteinMPNN:

• Designed Degraders combine de novo binders against conserved epitopes with engineered degrader domains to trigger target proteasomal degradation.
• Transcription Factor Modulators leverage de novo oligomers to pre-organize conserved DNA-binding domain motifs and outcompete endogenous transcription factors.
• Common Substrate Modulators use redesigned interaction domains (such as a fused Cullin5 subdomain and SOCS box domain) to sequester shared signaling substrates like Elongin B and C, thereby broadly inhibiting target family engagement.

Pan-NR4A and multi-family regulators dramatically boost T cell persistence and potency

Across yeast display and primary T cell screens, hundreds of de novo binders showed high affinity against target protein families. In repeated tumor co-culture assays using ROR1-targeted CAR-T cells, OUTLAST Regulators targeting NR4A, Cbl, and SOCS families restored cytotoxicity and cytokine production under conditions where conventional CAR-T cells became entirely dysfunctional. The pan-NR4A regulators achieved the strongest results overall, with the pan-NR4A Designed Degrader maintaining uninterrupted tumor-killing capacity across eight rounds of serial tumor challenges in vitro. Furthermore, flow cytometry revealed that pan-NR4A OUTLAST Regulators reduced exhaustion markers, such as TIGIT and PD-1, across both CD4+ and CD8+ T cell populations.

In vivo, inducibly expressed pan-NR4A Designed Degraders conferred 100% survival and robust tumor volume control in mice bearing ROR1+ H1975 human xenografts, while enhancing T cell proliferation and maintaining a naive/stem-like and central memory phenotype (CD45RA+/CD62L+). The approach also generalized beyond CAR-T cells: incorporating the pan-NR4A Designed Degrader into PRAME-targeted TCR-T cells reduced TIGIT expression, increased cytokine production, and established strong tumor control in a PRAME+ H226-A*02:01 lung carcinoma xenograft model.

Modular design platform opens new avenues for multiplexed cell and gene therapies

Since OUTLAST Regulators operate as compact genetic modules compatible with standard viral vectors and cell therapy manufacturing processes, they offer a versatile platform to program complex biological circuitry without permanent genome editing. Beyond ex vivo edited T cells, these heterobifunctional designs can readily extend to other cell types, regulated promoters, and gene therapy modalities to direct cell fate across diverse therapeutic applications.

“By considering biological phenomena as a structural problem, we can view them in a new way,” said Brian Weitzner, Head of Information Sciences at Outpace Bio and a Rosetta Commons board member. “In this case we identified a common surface across transcription factor family members and generated a binder specific to it using protein design. Solving real-world challenges like this is only possible due to advancements in protein design and we are only at the beginning of what we will achieve.”

Image inspired by Figures 3A and 3C in Boyken et al. (2026).